1. Field of the Invention
The present invention is directed to centrifuge tubes and associated sealing means for use with ultracentrifuge apparatus, and more particularly, relates to a thin wall, flexible centrifuge tube and associated sealing mechanism.
2. Description of the Prior Art
The need to place a liquid sample into a centrifuge rotor and subsequently remove it after centrifugal separation has caused the development of a wide variety of centrifuge containers with particular features suiting specific work. The purpose of a removable container, whether rigid, flexible, translucent or transparent has been to permit the operator to externally prepare a sample, load it into a rotor, perform centrifugation, remove the container with care to prevent remixing, and then to examine or analyze the separated sample.
With respect to ultracentrifugation where rotational speeds exceed 25,000 rpm, four primary categories of containers have gained acceptance and are in widespread use: rigid thick wall open-ended cylindrical tubes with hemispherical bottoms, flexible thin wall open-ended cylindrical tubes with hemispherical bottoms, rigid thick wall cylindrical bottles with flat or hemispherical bottoms, and flexible integral cylindrical tubes with essentially hemispherical tops and bottoms which are sealed by heat treatment rather than by mechanical cap or closure means applied in connection with the centrifuge process.
Rigid thick wall open-ended cylindrical tubes were developed primarily for fixed angle rotors where the angle of the tube with respect to the spin axis is less than 90 degrees; typically, the angle is 10 to 45 degrees. The rigid tube walls allow for a fill level low enough to prevent fluid spilling out during centrifugation since the walls are strong enough to withstand the centrifugal forces without containing a force-absorbing liquid. These tubes are generally reuseable because of their minimum residual distortion during centrifugation. The tube is unloaded from the open upper end and the walls or bottom need not be punctured or sliced as with flexible tubes to facilitate sample removal. In some cases, caps are used to increase the available volume of the tube by preventing spillage during centrifugation even with the increased fill volume. These tubes are generally costly and cannot be punctured by a needle for sample removal, and if hazardous biological samples require a single use and then discard of the tube, the cost is usually prohibitive. These tubes are used primarily for the convenience of quick loading of the liquid sample but at the disadvantage of a loss of available internal volume.
Flexible thin wall cylindrical tubes with open ends are used in swinging bucket rotors without caps. The angle of the tube with respect to the spin axis is 90 degrees and therefore no caps are required since the centrifugal forces acting on the fluid are directed towards and hold the fluid in the bottom of the tube. Because the tube is open at one end and the liquid meniscus is very near the top of the tube, tubes of larger diameter are subject to spillage by sloshing and wave action of the meniscus as the rotor with the tube in a bucket is placed in the centrifuge. Such spillage can actually facilitate tube collapse during centrifugation by loss of available side wall support or can contaminate the rotor if radioactive samples are used. Flexible thin wall tubes when used in fixed angle rotors require cap assemblies to contain the liquid under high hydrostatic pressure and to prevent collapse of the tube. Typically, the cap assemblies consist of relatively complex metal components and the primary seal is formed with the tube by an elastomer ring or bushing. In L. C. Marks, "Centrifuge Test Tube Cap", U.S. Pat. No. 3,459,369 a typical capping mechanism of this type is shown. As higher speed rotors have been developed, improvements in capping means have been required. Open-end tubes achieve their seal at the maximum diameter of the tube, and with lager diameter tubes, the cold flow potential of elastomeric seals increases, thereby making sealing more difficult. In H. E. Wright, et.al., "Capping Assembly for Thin Wall Centrifuge Tubes", U.S. Pat. No. 3,938,735, the elastomeric sealing ring at the large diameter of the tube is eliminated and a compression of the tube outer wall is utilized for sealing. High torques are required to compress the relatively large annular ring at the seal point and a low friction bushing is incorporated on the top of the crown to keep the crown from twisting with respect to the stem during the procedure to tighten the assembly. In the two patents of marks '369 and Wright '735 special bench-mounted vises and calibrated torque wrenches are required to achieve a reliable seal. Both also require an additional fill hole for either the entire sample addition or for "topping up" the tube after insertion of the main crown and stem. It is essential that the maximum volume of liquid be added so that collapse of the tube into empty regions is minimized during centrifugation. Also, the opaque metal components make it difficult to determine the exact fill level and the size of residual, trapped bubbles. For this reason, viewing holes are added to the side of the stem skirts, a feature which potentially weakens the thin skirt. Due to the inability to see trapped bubbles, complete filling of the tube is extremely difficult to achieve. The threaded fill hole must also be sealed, which is accomplished by the insertion of a small set screw which in turn compresses a small plastic bushing swaged in the bottom of the fill hole. After repeated use, the plastic bushing may extrude through the hole and settle in the previously filled tube. Another bushing must then be inserted, swaged with a special tool, and the set screw torqued into place. As a result, density gradient layers, carefully formed in the liquid contents of the tube, may be disturbed with the extended handling and torquing procedures. The approach described in L. Gropper, et al., "Centrifuge Test Tube Stopper", U.S. Pat. No. 3,720,502 has had no practical application.
Another application of flexible, thin wall cylindrical tubes with open ends is with vertical rotors where the angle of the tube with respect to the spin axis is zero. This application requires special consideration for the sealing of open-ended tubes because the maximum hydrostatic pressure coincides with the maximum diameter of the tube at the maximum spin radus, and the minimum pressure coincides with the diameter of the tube at the minimum spin radius. The pressure and gravitational force gradients across the tube diameter causes severe cold flow of the seal material and uneven distribution of initial cap torquing forces. In V. C. Rohde, "Method of Gradient Separation", U.S. Pat. No. 4,015,775 a zonal gradient separation method which makes use of this phenomenon is described although no tube or sealing form is disclosed. In S. J. Chulay, "Tube Cap Assembly for Preparative Centrifuge Rotors", U.S. Pat. No. 4,076,170 a complex metal and elastomeric sealing assembly for centrifuge tubes is described. In R. B. Anderson, "Dual Seal Arrangement for a Centrifuge Rotor Tube Cavity", U.S. Pat. No. 4,087,043 a metal and elastomeric seal assembly is described which also incorporates the technique of compressing the outer diameter of the tube wall inwardly as described above for Wright '735. Both require that the top hole be filled with a set screw and seal bushing so that they both possess the limitations described previously. In W. A. Romanauskas, "Centrifuge Tube Encloser", U.S. Pat. No. 4,114,803 the use of a large diameter swage plug in combination with a plastic liner to achieve a seal against the tube wall is described; the tube in turn is compressed directly against a seat at the top of the rotor cavity itself. Another embodiment provides a back-up ring in place of direct compression against the rotor seat. Problems with reliability of this seal led to the improvement of D. A. Webster, "Centrifuge Tube Encloser", U.S. Pat. No. 4,166,573 which provided grooves in the swage plug and claimed improved reliability. In D. A. Webster, et. al., "Centrifuge Tube Seal", U.S. Pat. No. 4,222,513 this general approach was further developed by adding an elastomer seal to maintain an axial force to thereby compensate for cold flow of the sealing components.
Rigid thick wall cylindrical bottles with threaded necks are primarily used for convenience. The bottles are sealed by the use of plugs fitted with elastomeric rings which seal against the internal or upper surface of the rigid neck. Threads on the outside diameter of the neck are present to provide means of applying compression to the elastomeric seals by means of a locking cap. Bottles like rigid open-ended tubes are costly, reuseable, unpunctureable and unslicable and sacrifice convenience for internal volume and easy sample removal. In N. Cho, "Centrifuge Sample Holder", U.S. Pat. No. 3,366,320 an older type of centrifuge bottle is described where no elastomeric seals are used and where a seal relies on a integral v-shaped ring to press against the top of the bottle neck. This sealing method cannot be used with the higher speed ultracentrifuge rotors (more than 25,000 rpm) because the cap and neck distort under the extreme forces and allow leakage. In W. J. Piemonte, et.al., "Bottle Support and Cap Assembly for Centrifuge", U.S. Pat. No. 3,071,316 a bottle sealing cap and support is described which minimizes distortion of the neck. The bottle extends beyond the rotor cavity to maximize the size of the bottle and an extending bottle neck support is required to maintain the neck geometry and therefore the seal. Although the bottles are described as distortable, they are in fact rigid in comparison with a thin wall, flexible centrifuge tube. In D. F. Mitchell, "Plastic Centrifuge Bottles and Caps Therefor", U.S. Pat. No. 3,265,296, and in A. J. Barletta, "Centrifuge Bottle and Closure Therefor", U.S. Pat. No. 3,434,615 rigid bottles sealed with elastomeric seals and screw caps are described.
Flexible, integral one-piece centrifuge tubes are used in both fixed angle and vertical rotors. Although caps as such are not required, support spacers with precise features are required to provide support to the upper end of the tube and to prevent collapse. The spacers are usually single piece plastic or metal shapes and do not contact the fluid directly. Although the integral tube and associated spacer appear less complex, the complexity has merely been shifted from a mechanical assembly within the rotor to an electro-mechanical apparatus on the laboratory bench. The sealing apparatus contains resistance heaters, timers, force applying arms, etc., and requires that holding blocks precisely made for each tube size, be exactly positioned under the heating unit; seal formers are required in addition to the heating and cooling apparatus to melt and mold the seal at the top of the tube. The integral single-piece tube also has the disadvantage that it can only be used once since the seal is formed from an integral stem which is generally cut off in the procedure of removing the sample after centrifugation. Because the integral stem is melted and formed to provide the seal, the diameter of the initial fill hole is kept small in an attempt to control the amount of molten plastic. The small fill hole unfortunately restricts access to the tube. Tube filling is therefore commonly accomplished with hyperdermic needles which must be inserted through the stem hole. The resultant fluid passage into the stem is very small and can cause the shearing of long, fragile molecular chains of biological compounds such as DNA. Unloading may be equally difficult. And, problems in heat sealing may occur when residual drops of fluid remain in the stem region. In addition, the subsequent melting of the plastic may vaporize the residual fluid producing sufficient local vapor pressure to cause the stem to reopen before solidification of the molten stem material. Since the only available plastic material is in the integral stem, additional attempts to reseal the tube often are not successful, and the tube must be discarded after inconvenient retrieval of the uncentrifuged sample. Although there are no metals present as in the other cap assemblies described above, the effects of heat may be detrimental to a sensitive biological sample. Integrable single piece tubes are described in the following patents: S. T. Nielsen, "Integral One Piece Centrifuge Tube", U.S. Pat. No. 4,301,963; S. J. Chulay, et.al., "Supporting Cap for Sealed Centrifuge Tube", U.S. Pat. No. 4,304,356 describing a spacer and integral tube with a bell-shaped top rather than the essentially hemispherical top to give greater rigidity to the spacer; S. J. Chulay, et.al., "Modular Supporting Cap and Spacer for Centrifuge Tubes", U.S. Pat. No. 4,290,550, describing a modular spacer to be used in conjunction with a bell-shaped tube top in both vertical and fixed angle rotors; and in K. Ishimaru, "Centrifuge Apparatus for Reorienting Gradients", U.S. Pat. No. 4,360,150 describing an integral one-piece tube with a spherical geometry to facilitate gradient reorientation.
In centrifuge tubes it is desirable that liquid samples be capable of being added to the tube before a cap or crown is in place so that it can be filled to an optimum volume and so that air bubbles can be examined and removed. It is also desirable to be able to readily obtain access to the liquid sample in the centrifuge tube before and after centrifugation. Preferable, after access is obtained the centrifuge tube can easily be resealed and reused. And it is desirable that a centrifuge tube be provided which can fit into ultracentrifuge rotors of different inner diameters. Also, a centrifuge tube is most useful when it can be used with vertical, angled rotors. All of the above objects are addressed by the centrifuge tube with removable crown and swage fitting of the present invention.